Probiotics research in Galleria mellonella

Keywords:

• Candida albicans
• Galleria mellonella
• infection model
• Lactobacillus
• Probiotic

Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” In essence, this definition was first coined in 2001 by an international expert panel convened by the Food and Agriculture Organization of the United Nations (FAO) and the WHO.¹ Validity and acceptance of the definition over the years is evidenced by the fact that only a minor grammatical correction was recommended in a recent expert panel meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP).² Clear definitions of probiotics are crucial for scientific research on probiotics, for producers and consumers of probiotic products, and for regulators who want to ensure product safety and protect the consumer from false or exaggerated health claims. Rigorous scientific research is necessary to prove or disprove that particular strains or groups of microorganisms confer health benefits to the host. The most recent recommendations on probiotics state that certain genera and species exert beneficial effects that can be considered probiotic.² For example, genera-level effects include competitive exclusion of pathogens, butyrate and other short chain fatty acid (SCFA) production, or regulation of intestinal transit. Probiotic species might produce vitamins or beneficial enzymes, metabolize bile salt, exhibit antagonisms toward specific pathogens, neutralize toxic chemicals or carcinogens, and support barrier function of the intestinal epithelium. Far reaching immunological, endocrinological, and neurological effects, however, are usually associated with specific strains of probiotics that are equipped with genetic traits that are not present in other members of the species. It remains to be determined whether the core benefits of probiotic genera/species are harbored in a “probiotic core genome” while strain-level probiotic mechanisms are encoded in highly specialized gene sets. At present, undefined microbial consortia (e.g. fecal microbiota transplants^3,4) that exert beneficial effects on the host are not considered probiotics according to the latest expert panel report². However, increased scrutiny of human or animal microbiota most likely will lead to identification of novel probiotics that are not members of the traditional genera with beneficial properties such as Lactobacillus or Bifidobacterium. For example, Faecalibacterium prausnitzi^5,6 and Akkermansia muciniphila^7,8 are considered as candidate probiotic species because of their positive effects on intestinal health in humans, but safety and efficacy of these bacteria has to be demonstrated through rigorous scientific research and randomized controlled trials. Consequently, selection and testing of microorganisms for potential use as probiotics in humans or animals is no easy task. In 2001 and 2002, expert consultations led to formulation of the FAO/WHO Guidelines for the assessment of probiotics in food.^1,9 These guidelines are still helpful today for selecting and evaluating probiotics as food, food supplement, or as probiotic drug for treatment and prevention of certain diseases.^2,10,11 While evaluation of safety and efficacy with suitable methods is paramount, appropriate viability and shelf-life are, by definition, prerequisites for probiotics. Ultimately, efficacy and safety have to be confirmed in the intended target organism, i.e. for human probiotics through well-conducted clinical studies in humans. In vitro studies of probiotic candidates or studies in model organisms can only provide an initial motivation for health claims, but no proof. Nevertheless, such research is a valid starting point and could give insights into potential mechanisms of probiotic action. Tests for tolerance to gastric acidity, resistance to bile, adherence ability to mucus and/or intestinal cells, and antimicrobial activities were suggested as in vitro studies by probiotics expert panels.^1,9 Many researchers have adopted these approaches to screen for potentially probiotic strains. In vivo studies using rodents, zebrafish (Danio rerio), or invertebrates such as Caenorhabditis elegans and Drosophila melanogaster as animal models are certainly better suited to deliver relevant information on probiotic mechanisms. For example, some of the most exciting research on the microbiota-gut-brain axis and potential manipulation of cognitive functions by probiotics has been conducted in mice.^12,13 The zebrafish model revealed the enhancement of fecundity and bone development by probiotic administration.^14,15 C. elegans has emerged as versatile model for the study of pathogens, commensals, and probiotics. ^16-19 Interestingly, symbiotic lactobacilli appear to be involved in metazoan intestinal development and stem cell proliferation, as recently reported in the fruit fly by Jones et al.²⁰

In this issue of Virulence, Vilela and coworkers²¹ add another invertebrate model system to the probiotic researchers’ tool box: the wax moth Galleria mellonella. While G. mellonella is not (yet) as genetically tractable as D. melanogaster or C. elegans, simplicity of manipulation and infection of G. mellonella larvae combined with their survival at 25 and 37°C, make the wax moth an attractive model system.²² Especially in research on bacterial^23,24 and fungal^17,25-29 pathogenesis, G. mellonella larvae have shown great promise as infection model. In their study, Vilela et al. utilized the larvae as model for infection with the pathogenic yeast Candida albicans and co-infection with a beneficial Lactobacillus acidophilus strain.²¹ Experimental candidiasis is well established in G. mellonella^30-32 and thus an attempt to ameliorate the lethal outcome of the fungal infection by administration of the probiotics is a logical approach to test for probiotic interference with C. albicans pathogenicity. The authors initiated their study by demonstrating anti-Candida effects of the lactobacilli in vitro, i.e., inhibition of fungal filamentation and biofilm formation. Vigorous production of lactic acid by lactobacilli has already been shown to exert antagonistic effects on filamentation and C. albicans cell viability, especially under unbuffered low pH conditions, despite the relatively high acid tolerance of these fungi.³³ Additional inhibitory substances produced by the lactobacilli might exacerbate these effects. Vilela et al developed an inoculation protocol for co-infection and confirmed that the lactobacilli were not pathogenic for the G. mellonella larvae. Subsequently, co-infection experiments revealed that the L. acidophilus strain indeed was able to reduce fungal burden in the larvae and lethality of the infection. The model system also allowed the researchers to perform histological analyses to characterize the effects of the beneficial bacteria on the fungal growth patterns in vivo. The work offers a first proof of concept, but many questions about the probiotic mechanisms encountered in this model remain open. How do the lactobacilli suppress C. albicans in the larvae? What is the role of the insect immune system? Are the findings relevant to probiotic research in humans?

In summary, a new invertebrate model to elucidate some aspects of probiotic interference with microbial pathogens has been introduced. The model system has clear advantages such as ease of use and reduced costs, especially in comparison to vertebrate models²². In contrast to D. melanogaster and C. elegans, pathogenic mechanisms of fungal or bacterial infections and host immune defense mechanisms in G. mellonella appear to correlate more closely with those encountered in humans.²² Conversely, the lack of a complete genome sequence, the absence of reporter and mutant strains, and the need for standardization across different sources and laboratories,^22,34 currently dampen the enthusiasm for the G. mellonella model; although future progress is likely to improve upon these drawbacks. In conclusion, G. mellonella has merit as a new model for foundational research on probiotics, but it will certainly not replace the randomized controlled trials in humans so urgently needed to evaluate efficacy of many so-called “probiotics."

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.